STUDIES FOR DETERMINING AND IDENTIFYING THE CONTENT OF ANTOCYANIC PIGMENTS IN SOME SPECIES OF WILD FRUITS ORIGINATED IN NEAMT COUNTY (2004)

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1 THE CONTENT OF ANTOCYANIC PIGMENTS IN SOME SPECIES OF WILD FRUITS STUDIES FOR DETERMINING AND IDENTIFYING THE CONTENT OF ANTOCYANIC PIGMENTS IN SOME SPECIES OF WILD FRUITS ORIGINATED IN NEAMT COUNTY (2004) S. COŞOFREŢ 1, D. BECEANU 2*, Roxana Mihaela RADU 2 1 Centre for Oenology Research, Iaşi Branch of Romanian Academy 2 University of Agricultural Sciences and Veterinary Medicine of Iaşi ABSTRACT The biological material analysed in this study has been sampled from different localities of the Neamţ County. In wild cherry and raspberry, the sampled harvesting was carried out at two different ripeness periods, for comparing the results. Wild blackberries contained a major pigment (cyanidine 3 glicoside) at a percentage of almost 90% of the whole antocyans present in fruits. Wild black cherries had three major characteristic pigments: peonidine-3-rutinoside (the highest percentage), followed by cyanidine-3-glucoside and peonidine-3-gluoside (the lowest percentage). In raspberry, we have identified the following pigments: cyanidine-3-sophoroside (major pigment) at a percentage of almost 50%, cyanidine-3-glucosidrutinosode and cyanidine-3-rutinoside. In blueberries, over 14 pigments could be found as distinct peaks. We have also identified the pigment cyanidine-3-glicoside, which was one of the most important. In case of blueberries, by comparing the own chromatogram to already published chromatograms and analysing the results, we could assess that main differences were found between the percentage of antocyans present in the cultivated varieties, respectively, the spontaneous flora. It also appeared the differentiation according to geographical area and species. Key words: wild fruit, antocyans, Neamţ County INTRODUCTION The theoretical, but especially the practical value of identifying the phenolic compounds is of present interest, this being illustrated by the numerous research teams approaching the subject nowadays (Harborne, 1988;. Hendry, * dumitru.beceanu@gmail.com 51

2 S. COŞOFREŢ ET AL. 1982). Knowing the types of available and cyanic compounds, one can estimate the pigment constancy, the technological qualities but particularly the curative properties of wild fruits. The biological material studied in the area of Neamţ County (Table 1) comes entirely from the perimeter under the administration of Neamţ Forest Department. Some species have been sampled from different places: wild cherries Piatra Neamţ, Tîrgu Neamţ; raspberry - Piatra Neamţ, Mitocu Balan-Cracaoan and Budecu; wild strawberry Piatra Neamţ; blackberry Cracaul Negru; blueberry - Poiana Mare and Budecu. In the case of rose hips and boxthorn, each sample comes from a different biotype, but in boxthorn, the identification of size, shape and fruit disposition on the branch was mentioned as well. In the case of wild cherries and raspberry, the harvesting was done at intervals, at two different ripening periods, in order to achieve a comparison of the results (Table 1). Biological material studied during one year (mean data) Table 1 Produce Wild cherries Medium samples Raspberry Medium samples Blackberry Medium samples Blueberry Medium samples Sampling date Place/zone/sample/characteristics III / June Fruit Delivery Centre, Piatra Neamţ V1.1, V1.2 I / July Fruit Delivery Centre, Tîrgu Neamţ V2.1, V2.2, V2.3 III / July Fruit Delivery Centre, Piatra Neamţ V1.1, V1.2 III / July Fruit Delivery Centre, Piatra Neamţ V2 I/ August V3.1, gathered in the field Mitocu Balan-Cracauan O.S.Varatec V3.2, V3.3, V3.4, gathered in P. Budecu, O.S.Borca, height 1300 m exposure N-E III/August Fruit Delivery Centre, Cracaul Negru Village, O.S. Varatec height 700m V1.1, V1.2 I / August V1.1, V1.2, gathered in the field P. Poiana Mare, O.S. Borca, height 1490m, exposure N-W V1.3, V1.4, gathered in the field P. Budecu, O.S.Borca, height 1300 m, exposure N-E Fresh fruit juice has been analysed, juice resulting of the following wild fruits: wild cherries, blueberries, raspberry, rose hips, boxthorn. All the juice samples have been limpid and stable. 52

3 THE CONTENT OF ANTOCYANIC PIGMENTS IN SOME SPECIES OF WILD FRUITS MATERIALS AND METHODS The Index of total polyphenols (ITP) or the D 280 Index represents the content of total phenolic compounds (phenolic acids, tanning materials and colouring matters) existing in the sample to be analysed. The method of determination (***, 1988; ***, 1990) is based on the property of benzene nuclei characteristic to phenolic compounds of completely absorbing the ultraviolet light, having a maximum of about nm. The juice obtained out of the fresh fruit is clarified by centrifugation or filtration, and then diluted to 1 per cent, using distilled water. The absorbent at 280 nm is read in the quartz vat of 1 cm (10 mm) optical line compared to the distilled water. Consider A 280 the optical intensity (the absorbent) read on the spectrophotometer. The Index of total polyphenols (ITP) or the D 280 Index equals: where d is the dilution factor of the initial solution. By means of a standard curve line, used for established concentrations of phenolic compounds, one can determine the concentration of phenolic compounds in g/l. The antocyan determination was done by sulphurous acid bleaching. The method is based on the antocyan property of reaction to the sulphurous acid in an acid medium thus forming colourless sulphite combinations. The distinction in optical intensity, read at 520 nm, between an unbleached sample and the same sample bleached by sulphurous acid is proportional to the antocyan content. The expression of antocyan content (g/100 g produce) is done according to a standard curve line. Antocyan identification in wild fruits. For a more complete characterization of wild fruits, an individual determination of antocyans was fulfilled by means of high pressure liquid chromatography. The obtained fresh juice was centrifuged and then filtered on the 0.45 µm porosity membrane. The chromatograms were recorded on a HPLC Hewlet-Packard 1100 binary pump, using a C18 column and a UV-VIS detector. The check reading was done at 546 nm. As an A eluent formic acid: water was used at 10:90 proportion and as a B eluent formic acid: acetone: water was used at 10:25:65 proportion, with increasing gradient of B eluent from 20 per cent to 70 per cent. RESULTS AND DISCUSSION The content in phenolic compounds of the analysed samples, expressed in g phenolic compounds/ 100g fruit, was presented in Table 2. Table 2 showed that the limits of phenolic compounds content in the wild fruits under analysis were between the maximal values of g/100 g analysed produce (blueberry) and g/100 g analysed produce (boxthorn). Important values were also signalled 53

4 S. COŞOFREŢ ET AL. in the case of blackberries (0.471 g/100 g produce) and of wild cherries (0.435 g/100 g produce). A more important content in total phenolic compounds can be also associated with high values of the quantities in vitamin P (biophlavone) in the produces under analysis (Beceanu et al., 2003; Gherghi et al., 1983; Gherghi et al., 2001). Table 2 Total content in phenolic compounds Species and order of content size Total phenolic compounds D 280 in phenolic compounds (g/100 g fruit) Wild cherries (III) Raspberry (V) Blackberry (II) Blueberry (I) Rose hips (IV) Boxthorn (VI) As for the antocyan content, expressed in g/100g produce, the variation limits (Table 3) were between 0.58 (blueberry) and 0.01 (boxthorn). By examining the comparative data, we could see that the order of size in total phenol compounds content was generally maintained. There was only one exception, namely wild black cherries, which exceeded the blackberries in antocyan content, occupying the second place. The reason would be the higher proportion of phenolic substances of antocyan type, compared to the blackberry. All the species with higher antocyan content had also higher values of colour components determined by means of CIE Lab 76 method. Total content in antocyans Table 3 Species and order of content size in antocyans Antocyan content (g/100g produce) Black cherries (II) 0.41 Raspberry (V) 0.09 Blackberry (III) 0.33 Blueberry (I) 0.58 Rose hips (IV) 0.12 Box thorn (VI) 0.01 By means of chromatograms obtained on HPLC, certain antocyans could be identified, while using the sulphurous acid bleaching method, a global content or a total quantity of the antocyans in the sample were determined 54

5 THE CONTENT OF ANTOCYANIC PIGMENTS IN SOME SPECIES OF WILD FRUITS After the chromatographic analysis on blackberry (Figure 1), one could draw the conclusion that the fruit prevailingly contained a major pigment (cyanidine-3-glicoside). This was identified at the retention time of 14.8 min (Figure 1). Calculating the area of the obtained peak, compared to the area of all the other concomitant peaks, we found that this pigment existed in a proportion of 90 per cent approximately out of the total antocyans present in the fruit. mau VWD1 A, Wavelength=518 nm (RUSU\MURE3.D) min Figure 1- The chromatographic analysis on blackberry In the case of wild black cherries (Figure 2), the achieved chromatogram indicates the presence of three peaks corresponding to the three major pigments characteristic to this species, namely: cyanidine-3-glucosidal, peonidine-3- gluoside and peonidine-3-rutinoside. Analysing the peaks we could notice that peonidine-3-rutinoside were in the highest proportion, followed by cyanidine-3- glucoside, and peonidine-3-glucoside, which were at the smallest quantity. In the case of raspberry (Figure 3), there was the possibility of chromatographic identification of the following pigments: cyanidine-3- sophoroside (at the retention time of 18.3 minutes), cyanidine-3- glucosidrutinosode (at the retention time of 19.8 minutes) and cyanidine-3- rutinoside (at the retention time of 21 minutes). Analysing chromatogram peaks we could notice that cyanidine-3- sophoroside was the dominant pigment, in a proportion of about 50 per cent. 55

6 S. COŞOFREŢ ET AL. Figure 2- The chromatographic analysis on wild black cherries mau 400 VWD1 A, Wavelength=518 nm (RUSU\ZMEURA2.D) min Figure 3- The chromatographic analysis on raspberry In the case of blueberries (Figure 4), a great variety of existing antocyanic pigments could be found, identifying thus over 14 such pigments under the form of distinct peaks. At the same time we could assert that, none of the existing pigments was dominant, noticing even a more or less equal presence of these components. The peak at the minute was successfully identified, belonging to the cyanidine-3 glicoside pigment, which was one of the most important, and corresponding to a proportion of about 12 per cent out of the total antocyan content. Compared to our own studies, in the studies already published 56

7 THE CONTENT OF ANTOCYANIC PIGMENTS IN SOME SPECIES OF WILD FRUITS (Harborne.,1988; Hendry and Houghton, 1982; Mazza, Miniati, 1993), the identification of a number of pigments similarly separated, and in the studies where different pigments were identified, those which were positively identified had a relatively small number. As far as the studied Actinium species concerned the research regarding Actinium corymbs L species (cultivated species) abound, where major differences could be found even among species. mau VWD1 A, Wavelength=518 nm (RUSU\AFIN_1.D) min Figure 4- The chromatographic analysis on blueberries CONCLUSIONS Blackberries contain a major pigment (cyanidine 3 glicoside) in a proportion of approximately 90 per cent out of the total antocyans present in the fruit. Wild black cherries have three major characteristic pigments: peonidine 3 rutinoside (in the highest proportion), followed by cyanidine 3 glucoside, and peonidine 3 glucoside which exists in the smallest quantity. In raspberry, there was the possibility of chromatographic identification of the following pigments: cyanidine-3-sophoroside (in a proportion of approximately 50 per cent), cyanidine-3-glucosidrutinosod and cyanidine-3- rutinosid. In blueberries over 14 pigments can be found under the form of distinct peaks more or less equal. The cyanidin-3-glicoside pigment, which is one of the most important, was successfully identified. 57

8 S. COŞOFREŢ ET AL. In the case of blueberries, comparing our own chromatogram to the chromatograms already published and then discussing the results, we can assert that there are major differences among the proportions of antocyans present in the cultivated species and in the spontaneous flora, respectively, also intervening the distinction according to the geographical area and species. REFERENCES Beceanu D, Coşofreţ S., Radu Roxana Mihaela, Rusu C., 2003 Etudes concernant la composition chimique de quelques fruits de bois recoltes de la flore spontanée du departement de Neamt. Scientific works. Series Horticulture, vol. 46, University of Agricultural Sciences and Veterinary Medicine of Iaşi Beceanu D, Coşofreţ S., Radu Roxana Mihaela, Rusu C., Etude sur les caracteristiques chromatiques de quelques especes de fruits sauvages provenus du departement de Neamt. Scientific works. Series Horticulture, vol. 46, University of Agricultural Sciences and Veterinary Medicine of Iaşi Beceanu D., 2003 Technology of horticultural products. Utilization in fresh estate and industrialization., Economica, Bucharest Cotea D.V., Sauciuc J.,1988 Treatise of Oenology, vol. 2. Ceres, Bucharest Favier J.Cl. et al., Repertoire general des aliments. INRA, Paris Gherghi A. et al., 1983 Biochemistry and physiology of vegetables and fruits. Academica Publishing House, Bucharest Gherghi A. et al., Biochemistry and physiology of vegetables and fruits. Publishing House of Romanian Academy, Bucharest Harborne J. B. (edited by),1988 The Flavonoids. Chamman and Hall Publishing House, London, 1988 Hendry, G.A.F and Houghton (edited by) 1982 Natural Food colorants. Blackie/Glasgow and London Luca C., Duca Al., Crişan I. Al., Analytical chemistry and instrumental analysis.didactica si Pedagogica Publishing House, Bucharest Mazza, G., Miniati, E.,1993 Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton, Ann Arbor London Tokyo Uzuru, Mihaela, 2000 New prospects of phytoterapeutical and phytocosmetic use of bush fruits (according to M. Melegari and Stefania Benvenuni, Rivista di Frutticoltura no ). Review Hortinform, no. 6/2000, Bucharest ***, (1988) Standard collection for vine industry. Ministry of Food Industry, Bucharest ***, (1990) - Recueil des methodes internationales d analyse des vins et des mouts. Office International de la Vigne et du Vin, Edition Officielle, Paris 58